CN113069534A - Application of IGSF10 in preparation of bone tissue regeneration products - Google Patents

Application of IGSF10 in preparation of bone tissue regeneration products Download PDF

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Publication number
CN113069534A
CN113069534A CN202110303273.3A CN202110303273A CN113069534A CN 113069534 A CN113069534 A CN 113069534A CN 202110303273 A CN202110303273 A CN 202110303273A CN 113069534 A CN113069534 A CN 113069534A
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bone
product
igsf10
tissue regeneration
bone tissue
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CN113069534B (en
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文晋
蒋欣泉
吴千驹
颜然
邓钰玮
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Priority to US17/376,191 priority patent/US20220296773A1/en
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    • A61K38/177Receptors; Cell surface antigens; Cell surface determinants
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Abstract

The invention relates to the field of medicines, in particular to application of IGSF10 in preparation of bone tissue regeneration products, and especially application of IGSF10 in preparation of bone tissue regeneration promoting products in combination with one or more growth factors of BMP, PDGF, EMP and amelogenin, wherein the products comprise periodontal bone defect repair products, jaw bone defect repair products and/or skull bone defect repair products. The new application of IGSF10 in the invention can reduce the use concentration of BMP, PDGF, EMP or amelogenin in the prior art and reduce adverse reactions, thereby exploring a method for promoting bone tissue regeneration and providing a new idea for treating bone defects.

Description

Application of IGSF10 in preparation of bone tissue regeneration products
Technical Field
The invention relates to the field of medicines, in particular to application of IGSF10 in preparation of bone tissue regeneration products.
Background
Regeneration of bone defects by tissue engineering is one of the research hotspots of researchers. Among them, growth factors play an important role in regulating cell proliferation, migration, extracellular matrix synthesis, and can effectively promote wound healing and bone tissue growth. The growth factors commonly used in clinic include morphological growth factors (BMPs) or mitogenic growth factors (PDGF), Enamel Matrix Proteins (EMP), amelogenin and the like, wherein the BMP family is an important growth factor known to promote differentiation of preosteoblasts and anterior cementum cells. However, adverse reactions such as swelling, ectopic mineralization and the like are often accompanied in the clinical application process of the factors, so that the search for growth factors which can be used alternatively or cooperatively to achieve bone tissue repair and the like becomes a current research hotspot.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of IGSF10 in the preparation of bone tissue regeneration products, for solving the problems of the prior art.
To achieve the above and other related objects, the present invention provides the use of IGSF10 in bone tissue regeneration.
Preferably, the application is the application of IGSF10 in bone tissue regeneration in combination with one or more of morphological growth factor (BMP), mitogenic growth factor (PDGF), Enamel Matrix Protein (EMP) and amelogenin.
The invention also provides application of IGSF10 in preparing bone tissue regeneration products.
Preferably, the application is the application of IGSF10 and one or more of morphological growth factors (BMPs), mitogenic growth factors (PDGF), Enamel Matrix Proteins (EMP) and amelogenin in preparing products for promoting bone tissue regeneration.
The invention also provides a product comprising a bone repair material and a growth factor loaded on the bone repair material, the growth factor comprising IGSF 10.
Preferably, the growth factors in the product further comprise one or more of BMPs, PDGF, EMP or amelogenin.
The invention also provides a method for repairing a bone defect, which comprises the following steps: administering the product to a patient having a bone defect.
As described above, the use of IGSF10 of the present invention in the preparation of bone tissue regeneration products has the following beneficial effects: the use concentration of BMP, PDGF, EMP or amelogenin is reduced, and adverse reactions are reduced, so that a method for promoting the regeneration of bone tissues is explored, and a new idea is provided for the treatment of bone defects.
Drawings
FIG. 1 shows the osteogenesis of DPC and PDLC by IGSF10 and BMP2 (A) alizarin red staining (B) calcium ion concentration detection.
FIG. 2 shows H & E histological examination of the repair effect of IGSF10 and BMP2 in a periodontal defect model.
FIG. 3 shows the Micro-CT effect of IGSF10 and BMP2 in repairing periodontal bone defects in rats.
FIG. 4 shows the effect of IGSF10 and BMP2 on Micro-CT repair of cranial defects in vivo.
Detailed Description
The present invention provides, first, the use of IGSF10 in bone tissue regeneration.
IGSF10(immunoglobulin superfamily member 10) is a member of the immunoglobulin superfamily and belongs to growth factors. In the present invention, the bone tissue regeneration includes cartilage tissue regeneration and bone tissue regeneration, unless otherwise specified. Regeneration refers to the process of repair by the division and proliferation of cells in adjacent healthy tissues after damage to the cells or tissues.
In particular, the use is the use of IGSF10 in the regeneration of bone tissue in mammals.
The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.
In one embodiment, the use is the use of IGSF10 for promoting bone tissue regeneration. IGSF10 can promote bone tissue regeneration by inducing and maintaining differentiation in early stages of bone or cartilage development.
The bone tissue is, for example, periodontal bone, jaw bone, skull bone, tibia bone, fibula bone, femur bone, or other bone tissue of the whole body system.
In a preferred embodiment, the use is IGSF10 for use in periodontal, jaw and/or skull defect repair.
The IGSF10 can be a native protein or a recombinant protein.
Further, the IGSF10 includes protein formed by any IGSF10 sequence from human, mouse, dog, cattle, pig and the like. The base protein sequences from different sources can be searched in NCBI. In one embodiment, the IGSF10 is selected from human recombinant IGSF10, having an amino acid sequence as set forth in SEQ ID No. 1:
SAFISPQGFMAPFGSLTLNMTDQSGNEANMVCSIQKPSRTSPIAFTEENDYIVLNTSFSTFLVCNIDYGHIQPVWQILALYSDSPLILERSHLLSETPQ(SEQ ID NO.1)
in a preferred embodiment, the use is the use of IGSF10 in combination with one or more of morphological growth factor (BMP), mitogenic growth factor (PDGF), Enamel Matrix Protein (EMP), amelogenin in bone tissue regeneration. After the combination, the use concentration of other growth factors can be reduced, thereby reducing adverse reactions.
In one embodiment, the BMP is selected from BMP-2.
In one embodiment, the concentration of each growth factor in the combination is 200-600 ng/mL. For example one selected from the following concentration ranges: 200-250 ng/mL, 250-300 ng/mL, 300-350 ng/mL, 350-400 ng/mL, 400-450 ng/mL, 450-500 ng/mL, 500-550 ng/mL, 550-600 ng/mL.
As a buffer for diluting the growth factors IGSF10, BMPs, PDGF, EMP or amelogenin, those skilled in the art will understand, and a commonly used buffer may be used as long as the properties of the growth factors themselves are not changed, such as physiological saline, PBS, Tris, etc.
The invention also provides application of IGSF10 in preparing bone tissue regeneration products.
In particular to the application of IGSF10 in preparing a mammal bone tissue regeneration product.
The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.
In one embodiment, the use is the use of IGSF10 in the preparation of a product for promoting bone tissue regeneration.
Such as periodontal bone, jaw bone, skull bone.
In a preferred embodiment, the use is the use of IGSF10 in the preparation of a periodontal bone defect repair product, a jaw bone defect repair product and/or a skull bone defect repair product. In one embodiment, is the use of IGSF10 in the preparation of a product for inducing and maintaining differentiation in early stages of bone or cartilage development.
The IGSF10 can be a native protein or a recombinant protein.
Further, the IGSF10 includes protein formed by any IGSF10 sequence from human, mouse, dog, cattle, pig and the like. The base protein sequences from different sources can be searched in NCBI. In one embodiment, the IGSF10 is selected from human recombinant IGSF10, having an amino acid sequence as set forth in SEQ ID No. 1:
SAFISPQGFMAPFGSLTLNMTDQSGNEANMVCSIQKPSRTSPIAFTEENDYIVLNTSFSTFLVCNIDYGHIQPVWQILALYSDSPLILERSHLLSETPQ(SEQ ID NO.1)
in one embodiment, the bone tissue regeneration product further comprises one or more of morphological growth factors (BMPs), mitogenic growth factors (PDGF), and Enamel Matrix Proteins (EMP) in addition to IGSF 10. That is, in one embodiment, the use is the use of IGSF10 in combination with one or more of BMPs, PDGF, EMP, or amelogenin in the preparation of a product for promoting bone tissue regeneration. The combination of the growth factors can reduce the use concentration of other growth factors, thereby reducing adverse reactions.
In one embodiment, the IGSF10 and/or other growth factors are used at a concentration of 200-600 ng/mL. For example one selected from the following concentration ranges: 200-250 ng/mL, 250-300 ng/mL, 300-350 ng/mL, 350-400 ng/mL, 400-450 ng/mL, 450-500 ng/mL, 500-550 ng/mL, 550-600 ng/mL.
As a buffer for diluting the IGSF10, BMPs, PDGF, EMP or amelogenin, it is known to those skilled in the art that a commonly used buffer can be used as long as the properties of the growth factor itself are not changed, such as physiological saline, PBS, Tris, etc.
Including but not limited to pharmaceuticals, nutraceuticals, foods, consumables, and the like. IGSF10 is the only active ingredient or one of the active ingredients of the product.
The product may be a single component material or a multi-component material.
In one embodiment, the product is a pharmaceutical product, which further comprises a pharmaceutically acceptable excipient.
By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.
Furthermore, pharmaceutically acceptable excipients should be compatible with the active ingredient, i.e. capable of being blended therewith without substantially reducing the efficacy of the medicament in the usual manner. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.
In another embodiment, the product is a consumable.
Specifically, the consumable can be formed by loading IGSF10 on a certain carrier alone or in combination with other growth factors. For example, IGSF10 and/or one or more of BMPs, PDGF, EMP or amelogenin are loaded onto bone repair materials to form the consumable. The bone repair material is a bridge for connecting seed cells and regenerated tissues, is structurally and functionally similar to natural bone, and has bone induction activity. The bone repair material is a hydroxyapatite bracket, a calcium phosphate bracket and a bioglass bracket.
The consumable can be prepared in the prior art, namely the growth factor and the bone repair material are independently stored, and the bone repair material is loaded with the growth factor when in use. The loading method comprises the following steps: slowly dripping the growth factors on the bone repair material, and transferring the bone repair material to a constant temperature incubator to stand for 1.5-2.5 hours for use. The temperature of the constant-temperature incubator is preferably 25-37 ℃.
In one embodiment, the growth factors include IGSF10 and BMP2, both supported on the bone repair material at a 1:1 ratio. Of course, the ratio between the growth factors can also be adjusted according to the specific experimental situation.
The invention also provides a bone tissue regeneration product, which comprises a bone repair material and growth factors loaded on the bone repair material, wherein the growth factors comprise IGSF 10.
In one embodiment, the growth factors in the product further comprise one or more of BMPs, PDGF, EMP or amelogenin.
In one embodiment, the bone repair material is a hydroxyapatite scaffold.
The preparation method of the bone tissue regeneration product is characterized by adding growth factors to a bone repair material, and standing for 1.5-2.5 hours at a constant temperature, wherein the growth factors comprise IGSF 10.
Specifically, the preparation method comprises the following steps: slowly dripping the growth factors on the bone repair material, and transferring the bone repair material to a constant temperature incubator to stand for 1.5-2.5 hours for use. The temperature of the constant-temperature incubator is preferably 25-37 ℃.
The growth factor also comprises one or more of BMP, PDGF, EMP or amelogenin;
the ratio between the growth factors was adjusted according to actual experiments.
The volume ratio of the total mass of the growth factors to the total mass of the bone repair material is 0.1: 1-15: 1, in ng: mm is3. For example, 0.1:1 to 1:1, 1:1 to 2:1, 2:1 to 5:1, 5:1 to 8:1, 8:1 to 11:1, or 11:1 to 15: 1.
The invention also provides a method for repairing a bone defect, which comprises the following steps: administering the bone tissue regeneration product to a patient having a bone defect.
Specifically, the bone tissue regeneration product is administered to a bone defect site of a bone defect patient.
The amount of growth factor in the product is such that a therapeutically effective amount is achieved. The "therapeutically effective amount" refers to an amount of a growth factor that is effective in treating bone defects, particularly periodontal bone defects, jaw defects, and/or skull defects.
In one embodiment, the therapeutically effective amount is 1ng to 160 ng. For example, the amount of the active ingredient is in a range of 1-10 ng, 10-20 ng, 20-70 ng, 70-120 ng, or 120-160 ng.
The bone defect refers to a bone shortage due to various causes such as trauma or surgery, and is called a bone defect. Due to the presence of bone defects, it is common to create nonunion, delayed or nonunion, and localized dysfunction of the bone.
The repair method also includes a series of conventional operations, such as anesthesia, skin incision, suturing, stitches removal, and the like.
The repair method further comprises treating the defect site with a substance prior to administration of the bone tissue regeneration product.
The repair method may be used in humans or other mammals.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
The recombinant IGSF10 protein used in the examples of the invention was purchased from Abcam, cat No.: ab 166199.
Example 1 alkaline phosphatase staining and calcium concentration detection
1.1 DPCs and PDLCs at 5X 104The cells were inoculated in 24-well plates at a density of one/mL, and incubated for 9 days after changing the medium, and then stained with alizarin red to observe calcium deposition.
1.2 harvesting the amount of cells required for each assay (2X 10)5Individual cells) were washed with cold PBS, resuspended in 500 μ L of calcium detection buffer and placed on ice. Blow up and down several times or use a homogenizer to homogenize the cells rapidly. Centrifugation of sample 2-5min to remove any insoluble material. Supernatants were collected and transferred to filters and samples were prepared in duplicate. All reagents were returned to room temperature. Setting a reaction hole: standard well 50 μ L standard diluent; sample wells 1-50 μ L sample (with dH)2O adjusted volume to 50 μ L/well). To each well containing the standard, sample and control, 90 μ L of color reagent was added. To each well was added 60 μ L of calcium assay buffer. Mix and incubate at room temperature for 5-10min in the dark. Measured on a microplate reader (OD575 nm).
As shown in fig. 1, alizarin red ARS staining shows that IGSF10 has strong ability to promote calcium nodule generation in the induction liquid environment, and comparison of the mineralization promoting effects of IGSF10 and BMP2 shows that IGSF10 is more significant than BMP2 in vitro, but the quantitative results show no statistical difference.
Example 2 rat periodontal Defect experiment
The surgical area was cleaned and disinfected with 75% ethanol and all surgical instruments were thoroughly cleaned and disinfected to minimize contamination. Anesthetized rats were induced with a combination of inhaled 4% (wt/vol) isoflurane and oxygen. The necessary dose of lidocaine was weighed out at 5mg/kg and injected subcutaneously in the back. After anesthesia of the rats, the nozzle supply of isoflurane was adjusted to 2.5% (wt/vol) maintenance. To prevent dryness, an eye ointment (lubricant) was applied to the rat eyes. After skin preparation in the surgical area, the skin was disinfected by alternate outward and spiral scrubbing with povidone-iodine topical preservative and sterile saline.
The skin is incised near the masseter at the lower edge of the mandible and extended posteriorly. The jaw bone and first molar area are seen after muscle dissection and separation, and after the distal root of the first molar and first molar exposure, a 3x2x1 mm defect is prepared using a round drill No. 4, removing the buccal root and cementum. IGSF10 was loaded on hydroxyapatite scaffolds at the same dose as BMP 2. The muscles were repositioned with absorbable nylon, the wound was closed and the skin was cleansed.
As a result, IGSF10 significantly promoted regeneration of bone tissue at the defect site with regular morphology, as shown in fig. 2 and 3; whereas the BMP2 group stimulated more strongly, the bone tissue exhibited expansive growth with morphologies that were irregular, and a condition similar to ectopic mineralization occurred.
Example 3 mouse skull Defect test
Male C57/BL6 mice were selected and weighed, and then anesthetized with isoflurane (5% for anesthesia induction and 1-3% maintenance of anesthesia status). The anesthesia status was monitored with the pinched hind limb and toe and the respiratory rate was observed at least every 5 min. The mice will be placed in the prone position and the surgical plate is incubated using a circulating warm water blanket. After shaving and disinfecting the skin, the mouse skull will be disinfected with 70% alcohol. The operator wears clean lab coat, headgear, mask and sterile gloves. Injection of 0.2mL lidocaine was performed at the incision site under local anesthesia. A2 cm long incision was made along the midline of the skull using a razor blade, the skin and periosteum were bluntly separated, and a circular bone defect was prepared on each side of the cranial suture, while cooling with saline, using 4mm od trephines.
In an in-vivo skull defect experiment, IGSF10 and BMP2 are loaded on a hydroxyapatite bracket in the same dosage, and the specific operation method comprises the following steps: according to the total mass of IGSF10 and BMP2 and the volume of the hydroxyapatite scaffold, the ratio of the total mass to the volume of the hydroxyapatite scaffold is 1.3: 1 (in ng: mm)3) Ratio of IGSF10 to BMP2 (R) using a pipette&D, usa) at 1; 1, transferring the mixture to a conventional incubator, standing for 2 hours, and loading the solution in the conventional incubator by using the siphonage effect of the porous structure of the hydroxyapatite scaffold for subsequent in vivo experiments. The wound was closed with 5-0 nonabsorbable thread after placing the different treated materials in the body. Removing the suture 10-14 days after the operation. All procedures were performed under sterile conditions. The materials are taken after 8 weeks, and micro-CT detection is carried out after 2 days of fixation by 4 percent paraformaldehyde.
As shown in fig. 4, IGSF10 and BMP2 loaded on hydroxyapatite scaffold at the same dosage both can effectively stimulate the formation of new bone, and the result of the synergistic loading of both shows that the skull defect part is completely replaced by new bone, thus it can be seen that IGSF10 greatly improves the osteogenesis effect of BMP 2.
The above results show that the IGSF10 is used for regeneration and repair of bone tissues, so that factors such as BMP which are clinically conventional at present can be synergized or replaced, the dosage of BMP can be reduced, adverse reactions can be avoided, and the clinical application of the BMP can be promoted to be feasible.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims (14)

  1. Use of IGSF10 in the preparation of bone tissue regeneration products.
  2. 2. Use according to claim 1, wherein said use is IGSF10 in the preparation of a mammalian bone tissue regeneration product.
  3. 3. The use according to claim 1, wherein the use is the use of IGSF10 in combination with one or more growth factors selected from BMP, PDGF, EMP, amelogenin for the preparation of a product for promoting bone tissue regeneration.
  4. 4. Use according to claim 3, wherein said IGSF10, BMP, PDGF, EMP or amelogenin is selected from a natural protein or a recombinant protein.
  5. 5. Use according to claim 1, wherein the product comprises a periodontal bone defect repair product, a jaw bone defect repair product and/or a skull bone defect repair product.
  6. 6. Use according to claim 1, wherein the product is selected from a pharmaceutical product, a nutraceutical product, a food product or a consumable.
  7. 7. A bone tissue regeneration product comprising a bone repair material and a growth factor loaded onto the bone repair material, the growth factor comprising IGSF 10.
  8. 8. The bone tissue regeneration product of claim 7, wherein the growth factors in the product further comprise one or more of BMP, PDGF, EMP, or amelogenin.
  9. 9. Bone tissue regeneration product according to claim 7, characterized in that said bone repair material is a hydroxyapatite scaffold, a calcium phosphate scaffold or a bioglass scaffold.
  10. 10. The preparation method of the bone tissue regeneration product is characterized by adding growth factors to a bone repair material, and standing for 1.5-2.5 hours at a constant temperature, wherein the growth factors comprise IGSF 10.
  11. 11. The method of claim 10, further comprising one or more of the following features:
    1) the growth factor also comprises one or more of BMP, PDGF, EMP or amelogenin;
    2) the constant temperature is 25-37 ℃;
    3) the ratio of the total mass of the growth factors to the total mass of the bone repair material is 0.1: 1-15: 1.
  12. 12. a method of repairing a bone defect, the method comprising the steps of: administering a bone tissue regeneration product according to any one of claims 7 to 9 to the site of a bone defect.
  13. 13. Repair method according to claim 12, characterized in that the bone tissue regeneration product according to any one of claims 7-9 containing an effective amount of growth factors is administered to the site of the bone defect.
  14. 14. The repair method of claim 12, wherein the repair method is useful for repairing a bone defect in a mammal.
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